There is considerable interest in proteins, as therapeutics and as biochemical and chemical reagents. However, most proteins are inherently unstable and degrade upon storage, transport and use, necessitating regulated temperatures, controlled solvation, and the addition of carrier molecules that may need to be removed. Proteins are also known to denature due to physical or chemical stresses such as desiccation, heat, light, and pH change, further complicating application of certain biomolecules.
While attachment of poly(ethylene glycol) (PEG) to proteins has been widely used to increase in vitro and in vivo stability for therapeutic proteins by reducing access of proteolytic enzymes and screening through the renal filtration systems, PEGylation alone does not normally significantly increase protein stability with regard to temperature, desiccation, and storage.
In nature, many plants and animals endure complete dehydration stress by accumulating large amounts of sugar. For example, alpha, alpha-linked glucose disaccharides have been known to impart unusual stability to organisms tolerating anhydrobiosis (desiccation) and cryobiosis (low temperature) by protecting cells and proteins. However, there is a need in the art for agents that are more effective at stabilizing and protecting biomolecules against degradation upon exposure to environmental stresses over extended periods of time.
Trehalose has been described as an excipient in therapeutic protein formulations. US Patent (U.S. Pat. No. 6,991,790) describes use of trehalose and other disaccharides along with surfactants to stabilize antibody formulations for up to two years. In particular anti-CD20 was targeted for use in treatment of B cell lymphoma. U.S. Pat. No. 6,821,515 summarizes trehalose used at a high ratio (100-600:1) to stabilize proteins or antibodies to lyophilization. In particular anti-HER2 may be reconstituted with minimal loss of activity for the treatment of breast cancer. Trehalose has also been described to increase the solubility of polypeptides in aqueous solutions including aqueous solutions that contain organic solvents.
Trehalose has been employed in solid dosage/tablet applications. U.S. Pat. Nos. 6,589,554, 7,074,428 and 7,575,762 describe use of trehalose as a binder for solid quick disintegrating tablets, as well as for sustained release, for use in the buccal cavity fabricating solid formulations of varying release times and hardness. U.S. Pat. Nos. 7,425,341, 6,740,339, 6,455,053, and 5,762,961 disclose trehalose used as a binder or diluent in the composition of a solid quick-dissolve tablet sometimes along with other polyols such as cellulose derivatives. U.S. Pat. Nos. 5,958,455 and 6,194,001 summarize general solid tablet formulation with trehalose and the formulation for an amoxicillin tablet.
Trehalose was used as an excipient in a spray freezing process for the production of solid tablets (U.S. Pat. No. 4,762,857) and was also used in a powder that is administered by inhalation (U.S. Pat. No. 7,785,631). Trehalose changes the taste of a bitter-tasting active ingredient (U.S. Pat. No. 6,998,139).
Trehalose has been used to preserve whole cells, tissues, or organisms in the following patents: Trehalose was described to stabilize eukaryotic cells that have been specially immobilized on a support matrix for cryopreservation (U.S. Pat. No. 7,314,755). U.S. Pat. Nos. 7,169,606 and 6,528,309 summarize blood platelets that are stabilized to cryopreservation via trehalose that is introduced with various methods, temperatures and pressures. U.S. Pat. Nos. 6,770,478 and 4,806,343 summarize red blood cells and proteins in artificial blood preserved by addition of trehalose with or without metal ions prior to lyophilization. U.S. Pat. Nos. 7,270,946, 6,528,309, and 5,827,741 disclose methods for stabilizing mammalian cells to lyophilization and cryopreservation respectively by using trehalose as an excipient. Bacteria have been preserved using trehalose in the medium (U.S. Pat. No. 6,610,531). U.S. Pat. Nos. 6,475,716 and 6,653,062 describe preservation of whole organs with trehalose and a trehalose preservation solution generally applicable to biologics, respectively. Trehalose combined with biologically safe acids or ethanol was used to prolong the shelf life of various products including pharmaceuticals (U.S. Pat. No. 6,005,100).
Trehalose has also been described in various administrations and deliveries. Examples are in the treatment of osteoporosis (U.S. Pat. No. 6,440,446), in the treatment of articular failure or to improve blood circulation (U.S. Pat. Nos. 7,214,667 and 5,981,498), in ophthalmic use (U.S. Pat. No. 6,555,526), in peptide or protein controlled release from glassy trehalose (U.S. Pat. No. 6,187,330), and in delivery of trehalose particles to cells (U.S. Pat. No. 5,840,878).
Previously, trehalose-based materials have been produced as cross-linked polymer networks including poly-substituted trehalose vinylbenzyl ether thermo-set resins (Teramoto and Shibata, 2004). Achieving trehalose linear polymers are challenging as the anomeric centers are relatively unreactive due to the 1,1-glycosidic linkage (Wolfenden and Yuan, 2008). Therefore, typical synthetic routes to produce trehalose-based monomers contain several protecting and deprotecting steps either by using bifunctional monomers targeting the 6,6′-positions, or by producing mixtures of regio-isomers that are not well defined. For example, a simple strategy for synthesizing trehalose linear polymers was first reported in 1979, but selectivity to form linear polymer versus the branched one was unclear at that time (Kurita, Hirakawa, et al., 1979).
Polymerization of diamino-type trehalose was explored to overcome the issue of branching, but the overall process was more complicated (Kurita, Masuda, et al., 1994). Acetalization (Teramoto, Arai, et al., 2004), enzymes (Park, Kim, et al., 2000), Diels-Alder reactions (Teramoto, Arai, et al., 2006), and click chemistry (Srinivaschari, Liu, et al., 2006 and 2007) have been exploited to synthesize trehalose-based linear polymers, with the later study being extended to biological systems. However, previous research and patents described reactions of incorporating trehalose into the polymer backbone rather than as a side chain. Polymers with trehalose in the backbone cannot be prepared in such a way to have end groups for attachment to biomolecules. Moreover, polymers with trehalose in the backbone cannot be prepared with narrow molecular weight distributions. Further, because the alcohols are important for hydration and protective properties, they may not protect biomolecules as well as a linear side chain polymer.
Although Kitagawa and his co-workers described a trehalose side chain polymer (Kitagawa, Chalermisrachai, et al., 1999), the polymers were synthesized by enzymatic synthesis. Enzymatic synthesis is extremely difficult to scale up. The polymers were prepared by free radical polymerization, and the reaction did not allow for the synthesis of one reactive end group for conjugation to biomolecules and the reaction did not prepare polymers with narrow molecular weight distributions. Moreover, the monomers were synthesized with excess divinyl adipates and any as-formed bis-functionalized trehaloses were not removed. Therefore, the as-prepared polymers were likely to be a mixture of products, containing cross-linked materials. Finally, the polymers were not used to stabilize biomolecules nor were they conjugated to proteins or other biomolecules.
A monomer was described in US patent of U.S. Pat. No. 5,856,416 with a the goal of obtaining cross-linked networks for use in contact lenses. Further, in U.S. Ser. No. 12/134,556 trehalose condensation polymers were claimed to stabilize biologically active ingredients, in particular nucleic acids in mixtures. The polymers contain trehalose as part of the backbone. Moreover, condensation polymers described in the patent application could not be prepared with end groups to conjugate to proteins and they can not be prepared with narrow molecular weight distributions.
PEGylation or attachment of polyethylene glycol (PEG) or PEG side chain polymers is known to enhance the pharmacological properties, e.g., protecting biomolecules from enzymatic degradation (Lyczak and Morrison, 1994; Syed, Schuyler, et al., 1997; Cohen, Yoshioka, et al., 1991). PEGylation alone does not typically significantly increase protein stability to temperature, desiccation, and storage. It has been recently reported that poly(carboxybetaine), a polyzwitterion, can be attached to proteins to enhance protein thermal stability (Keefe and Jiang, 2012; Yang, Zhang, et al., 2009). Recently, Applicants disclosed poly(stryrene sulfonate)-based polymers that stabilize heparin binding proteins to environmental stressors (PCT/US12/66905; Nguyen, Kim, et al., 2013).
Needed in the art is a homopolymer or copolymer with side chain trehalose having desirable protection capabilities.